Most cell types are highly sensitive to their physical environment and physiological forces acting on them play a dominating role in many regulatory cell functions. Such mechanotransduction is always studied using monotonous mechanical stimuli;however, cells in the body are exposed to irregularly varying stimuli. Two examples include breathing and circulation. Recently, we found evidence that in alveolar epithelial type II cells, both in culture and in vivo, the presence of physiological variability in stretch fundamentally alters the secretory response of these cells. Cells sense external mechanical forces via adhesion molecules and the cytoskeleton (CSK). It is thus feasible to assume that through the CSK, most if not all basic cell functions would also be sensitive to variability in mechanical stimuli. Evolutionary forces should favor structures that can adapt to and take advantage of existing variability. Accordingly, our central hypothesis is that physiological levels of variability in mechanical stimuli that are normally present in the body have fundamental regulatory roles in many basic cell functions. This aspect of mechanotransduction has been overlooked in cell and tissue culture studies. A major challenge is therefore to establish whether our findings are specific to epithelial cells or the phenomenon is general representing a major paradigm shift in mechanobiology. To test this hypothesis, we will use four different in vitro cell systems: lung epithelial cells, vascular endothelial and smooth muscle cells and skin or lung fibroblasts. We will test various outcomes in these cell systems in vitro while gradually changing variability in mechanical stimuli. Specifically, we aim to determine the effects of variable stretch (VS) pattern on transcription, translation and secretion of specific cytokines, enzymes and structural ECM proteins. We will also assess the effect of VS on basic cell functions such as division, growth and apoptosis to uncover universal mechanisms among different organ systems. Finally, to determine the possible effects of VS on metabolism, we will assess the generation of reactive oxygen species (ROS) during VS. To shed light on the mechanisms of VS-induced phenomena, we will employ various inhibitors along the mechanotransductory pathway during VS while imaging the constituents and organization of the CSK. We will then develop novel network models of the CSK to better understand mechanical force transmission from adhesion sites through the CSK to the nucleus. If our hypothesis is correct, then besides many basic cell functions, the production and secretion of enzymes, cytokines and ECM building blocks will all be affected by VS. Additionally, VS may also influence ROS which play a crucial role in the pathogenesis of several major diseases including atherosclerosis, neuro- degenerative diseases, metabolic disorders, aging and cancer. Thus, our project - the first to apply VS patterns to probe cell functions - could have far reaching transformative implications for the understanding of how cells work in real living tissues and hence for biology and medicine. This research could thus influence the way scientists including biologists, physiologists, physicists as well as clinicians think about the cell. PUBLIC HEALTH RELEVANCE: The basic functions of many cell types are sensitive to physiological levels of mechanical forces acting on them and this phenomenon is always studied in the laboratory using monotonous mechanical stimuli. However, cells in the body are exposed to irregularly varying stimuli and this variability may fundamentally alter all essential cell functions including secretion, growth and death. Uncovering how cells deal with such physiological variability may help understand how cells work in real living tissues as well as the pathogenesis of several major diseases including atherosclerosis, neuro-degenerative diseases, metabolic disorders, aging or cancer.